Recombinant human cytomegalovirus containing foreign gene

ABSTRACT

The present invention provides a virus comprising a DNA sequence essential for replication of a human cytomegalovirus and at least one foreign DNA sequence adapted for expression in a host. The foreign DNA sequence may encode a human immunodeficiency virus anti-sense mRNA sequence or an antigenic polypeptide, e.g., a malarial surface antigen. 
     Also provided are therapeutic compositions and vaccines which comprise the novel viruses of the present invention.

This is a continuation of application Ser. No. 067,375, filed Jun. 26,1987, now abandoned.

BACKGROUND OF THE INVENTION

The invention described herein was made in the course of work underGrant Nos. N43-AI-5-2603 and 1 R43-AI/CA24069-01-SSS from the NationalInstitute of Health, U.S. Department of Health and Human Services, andunder contract No. DAMD17-87-C-7046 from the Department of the Army. TheU.S. Government has certain rights in this invention.

Within this application several publications are referenced by Arabicnumerals within parentheses. Full citations for these references may befound at the end of the specification immediately preceding the claims.The disclosures of these publications in their entirety are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

Human cytomegalovirus (HCMV) belongs to the animal herpesvirus group.The cytomegaloviruses occupy a special subfamily of the Herpesviridaecalled the Betaherpesvirinae. Viruses belonging to this group exhibitnarrow host range, long duration of the replication cycle with slowlyprogressing lytic foci in cell culture, and frequently show enlargedcells (cytomegalia) both in vitro and in vivo. The viruses also have agenomic organization (an arrangement of genes) which is distinct fromother herpesvirus groups. These characteristics show thatcytomegaloviruses are distinct from herpesviruses in other subfamilies(1). Accordingly, what is known and described in the prior art regardingother herpesvirus subfamilies does not pertain a priori to members ofthe cytomegalovirus subfamily.

HCMV occurs widely in the human population, e.g., approximately 60% ofhumans have serological evidence of infection by HCMV by adulthood. Theinfections occur through direct transmission by contact, often throughmother's milk. The infection is usually asymptomatic, and HCMV has areputation for being a very benign pathogen. Serious complications arisein two instances. Fetuses that are infected in utero by passage of HCMVfrom the mother (who herself is suffering a primary infection) canbecome infected and be born with "cytomegalic inclusion" disease. Thisis a serious HCMV infection, indicated by the excretion of large amountsof HCMV in the urine, and often leads to congenital defects involvingsensorineural loss, e.g., hearing or psychomotor development, andretardation. The other instance involves any immunosuppressedindividual, e.g., someone on immunosuppresive drugs, an individualsuffering a congenital immunodeficiency, or an individual suffering anacquired immunodeficiency, e.g., AIDS. Because of the widespreadpresence of HCMV, these individuals frequently suffer from a generalizedHCMV infection which they are unable to combat immunologically. Thesetwo instances of HCMV diseases contribute a relatively large diseaseburden for which there is little or no therapy (2).

Two different HCMV strains have been tested as vaccines. The first testemployed the AD-169 strain of HCMV given to healthy volunteers asdescribed in (3). The test was successful with transient development ofcomplement-fixing antibodies and little untoward side effects except adelayed local reaction at the site of injection. The virus was againtested by another group who confirmed the previous results and showedthat immunological responses were maintained for at least one year (4).In an independent vaccine effort, the Towne strain of HCMV was grown intissue culture for 125 passages (Towne-125) and tested as a candidatelive virus vaccine (5). The results of the Towne-125 administrationparalleled those of AD-169 administration; antibody responses wereevident in healthy human volunteers, and a local delayed reactionoccurred at the site of injection (6, 7). A prospective study wasundertaken with the Towne-125 strain in patients with end-stage renalfailure who were candidates for transplants and hence immunosuppressivetherapy (8). All seronegative vaccinates developed antibody and none hadadverse side-effects except for local reactions, and no vaccine-relatedproblems were identified in the interval after transplantion. Howeversix of the nine vaccinates did excrete HCMV in their urine aftertransplantation and immunosuppressive therapy, albeit this was wild typeand not vaccine strain. Thus the vaccination did not protect fully,against infection or re-activation of HCMV, but statistically thepatients were better off for having been vaccinated. In a direct safetycomparison between the Towne-125 strain and a wild type Toledo-1 strainin healthy seronegative volunteers, the Toledo-1 strain was shown toinduce laboratory abnormalities or mild mononucleosis only, while theTowne-125 strain showed no adverse effects except for delayed localreactions at the site of injection (9). These studies show that existingstrains of HCMV have a high degree of safety, even in immunosuppressedpatients, which may be improved by reducing the delayed reactions at thesite of injection.

The present invention concerns the use of HCMV as a vector for thedelivery of vaccine antigens and therapeutic agents to humans. Thefollowing properties of HCMV support this rationale: HCMV is ubiquitousin nature; HCMV has benign effects in healthy individuals; an HCMVstrain exists which appears safe for immunocompromised individuals; andthe target population for an HCMV-delivered therapeutic agent is likelyto have been exposed to wild type HCMV and therefore should not have anincreased risk burden from the vector. Accordingly an attenuated HCMV isan excellent candidate for a viral vector delivery system, having littleintrinsic risk which must be balanced against the benefit contributed bythe vector's vaccine or therapeutic properties.

The prior art for this invention stems first from the ability to cloneand analyze DNA while in bacterial plasmids. The techniques that areavailable for the most part are detailed in Maniatis et al. (10). Thispublication teaches state of the art general recombinant DNA techniques.

Among the herpesviruses, only four herpesviruses (herpes simplex ofhumans, herpes saimiri of monkeys, pseudorabies virus andvaricella-zoster virus) have been engineered to contain foreign DNAsequences previous to this disclosure. The earliest work on the geneticmanipulation of herpes simplex involved the rescue of temperaturesensitive mutants of the virus using purified restriction fragments ofDNA (11). This work did not involve cloning of the DNA fragments nor thepurposeful creation of deletions nor insertions of foreign DNA fragmentsinto the viral genome. The first use of recombinant DNA to manipulateherpes simplex virus involved cloning a piece of DNA from the L-Sjunction region into the unique long region of the DNA, specificallyinto the thymidine kinase gene (12). This insert was not a foreign pieceof DNA, rather it was a naturally-occurring piece of herpesviruses DNAthat was duplicated at another place in the genome. This piece of DNAwas not engineered to specifically express any protein, and thus it didnot teach how to express protein in herpesviruses. The manipulation ofherpes simplex next involved the creation of deletions in the virusgenome by a combination of recombinant DNA and thymidine kinaseselection. The first step was to make a specific deletion of thethymidine kinase gene (13). The next step involved the insertion of thethymidine kinase gene into the genome at a specific site, and then thethymidine kinase gene and the flanking DNA at the new site were deletedby a selection against thymidine kinase (14). In this manner herpessimplex alpha-22 gene has been deleted. (14). In the most recentrefinement of this technique, a 15,000 bp sequence of DNA has beendeleted f rom the internal repeat of herpes simplex virus (15).

The insertion of genes that encode protein into herpesviruses haveinvolved a number of cases: the insertion of herpes simplex glycoproteinC back into a naturally occurring deletion mutant of this gene in herpessimplex virus (16); the insertion of glycoprotein D of herpes simplextype 2 into herpes simplex type 1 (17), again with no manipulation ofpromoter since the gene is not really "foreign"; the insertion ofhepatitis B surface antigen into herpes simplex virus under the controlof the herpes simplex ICP4 promoter (18); and the insertion of bovinegrowth hormone into herpes saimiri virus with an SV40 promoter that infact didn't work in the system (an endogenous upstream promoter servedto transcribe the gene) (19). Two additional cases of foreign genes(chicken ovalbumin gene and Epstein-Barr virus nuclear antigen) havebeen inserted into herpes simplex virus (20), and glycoprotein X ofpseudorabies virus has been inserted into herpes simplex virus (21).

More recently, the herpes simplex virus TK gene and the tissueplasminogen activator gene have been inserted into pseudorabies virus(PCT International Publication No. W087/00862), and an Epstein-Barrvirus glycoprotein antigen has been inserted into varicella-zoster virus(22).

These examples of insertions of foreign genes into herpesviruses do notinclude an example from the cytomegalovirus subfamily. Thus they do notteach methods to genetically engineer cytomegaloviruses, i.e., where tomake insertions and how to get expression.

The idea of using live viruses as delivery systems for antigens has arelatively long history going back to the first live vaccine. Theantigens were not "foreign" but were natural components of the livevaccines. The use of viruses as a vector for the delivery of "foreign"antigen in the modern sense became obvious with the vaccinia virusrecombinant studies. There vaccinia was the vector and various antigensfrom other diseases were the "foreign" antigens, and the vaccine wascreated by genetic engineering. While the concept became obvious withthese disclosures, what was not obvious was the answer to a morepractical question of which are the best candidate virus vectors. Inanswering this question, details of the pathogenicity of the virus, itssite of replication, the kind of immune response it elicited, thepotential for it to express foreign antigens, its suitability forgenetic engineering, its probability of being licensed by the FDA, etc,are all factors in the selection. For example, a viral vector carrying atherapeutic agent needs to target the correct cell type to deliver thetherapeutic agent. The prior art does not teach these utility questions.

Furthermore, the obvious use of vaccinia virus to carry foreign antigensdoes not extend to its use in the delivery of therapeutic agents.Moreover, the use of any herpesvirus as a vector for therapeutic agentshas equally not been pursued in the prior art.

The prior art relating to the use of viruses as therapeutic vectorsinvolves members of the retrovirus family. These viruses are distinctivebecause they integrate into the host cell genome during infection, andthey can be engineered to deliver foreign genes that potentially couldcure genetic diseases. This concept involving retroviruses cannot beextended to any other virus family by analogy because of the uniquenature of the retrovirus replication cycle.

The nature of the therapeutic agent that is to be delivered by a viralvector of the present invention must be a biological molecule that is aby-product of cytomegalovirus replication. This limits the therapeuticagent in the first analysis to either DNA, RNA, or protein. There areexamples of therapeutic agents from each of these classes of compoundsin the form of anti-sense DNA, anti-sense RNA, interferon-inducingdouble stranded RNA, and numerous examples of protein therapeutics, fromhormones, e.g., insulin, to lymphokines, e.g., interferons andinterleukins, to natural opiates. The discovery of these therapeuticagents and the elucidation of their structure and function does not makeobvious the ability to use them in a viral vector delivery system.

SUMMARY OF THE INVENTION

The present invention provides a virus comprising a DNA sequenceessential for replication of a human cytomegalovirus and at least oneforeign DNA sequence adapted for expression in a host.

The present invention also provides the novel plasmids designatedpSY1157 and pSY1159.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the HCMV (Towne) genome organization. An XbaI restrictionenzyme map, with cloning and insertion strategy illustrated, is alsoshown.

Legend:

L=long segment of the genome

S=short segment of the genome

J=junction

B=BamHI

E=EcoRI

P=PstI

S=SacI

X=SbaI

Xh=XhoI

IE=site of the immediate early promoter

↑=restriction sites targeted as potential insertion site

↑=restriction sites suitable for insertion for foreign genes

FIG. 2 schematically shows the insertion of PRVgpX/beta-gal into theEcoRI H fragment of Towne HCMV at the XhoI deletion, to generatehomology vector pSY920. Also shown is the insertion of HCMV IE/beta-galinto the EcoRI H fragment of Towne HCMV at the XhoI deletion to generatehomology vector pSY930.

Legend:

L=long segment

S=short segment

J=junction

FIG. 3 is made up of FIGS. 3A, 3B and 3C. FIG. 3A shows the HCMV (Towne)genome organization and XbaI, HIndIII, and EcoRI restriction enzymemaps.

Legend:

L=long segment

S=short segment

J=junction

FIG. 3B shows the confirmation of S-HCMV-001 structure by Southern blotanalysis. Lanes 1 and 7 contain molecular weight markers. Lane 2represents HCMV Towne DNA digested with HindIII and probed with XbaI MDNA. Lane 3 and half of Lane 4 represent the HCMV/beta-gal recombinant(S-HCMV-001) digested with HindIII and probed with beta-gal DNA. Lane 6represents HCMV Towne DNA digested with HindIII and probed with beta-galDNA.

FIG. 3C shows the confirmation of recombinant virus (beta-gal insert atthe XhoI deletion) structure by Southern blot analysis. Lane 1 containsmolecular weight markers. Lane 2 represents plasmid pSY1112 digestedwith EcoRI and probed with plasmid pSY844. Lane 3 represents HCMV TowneDNA digested with EcoRI and probed with beta-gal DNA. Lane 4 representsHCMV/beta-gal recombinant virus digested with EcoRI and probed withbeta-gal DNA.

FIG. 4 schematically shows the cloning of the 2.1 kb PstID fragment andthe 1.1 kb PstI to SacI fragment from Towne HCMV.

Legend:

L=long segment of the genome

S=short segment of the genome

J=junction

P=PstI

S=SacI

FIG. 5 schematically shows the insertion of PRV gpx/beta-gal into the5.4 kb BamHI fragment of Towne HCMV at the RI site to generate homologyvector pSY1112.

Legend:

L=long segment of the genome

S=short segment of the genome

J=junction

B=BamHI

E=EcoRI

X=XbaI

FIG. 6 (made of up FIGS. 6A and 6B) shows the insertion of the HCMV IEPstI to SacI fragment at the cap site of the HSV TK segment, to generatethe HCMV IE/beta-gal construct pSY1132. The DNA and amino acid sequencesof part of this fusion are also shown.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a virus comprising a DNA sequenceessential for replication of a human cytomegalovirus and at least oneforeign DNA sequence adapted for expression in a host. In one embodimentof the invention, the DNA sequence essential for replication of thehuman cytomegalovirus is derived from a naturally-occurring humancytomegalovirus. In another embodiment of the invention, the virus isattenuated. Furthermore, the foreign DNA sequence adapted for expressionin a host may encode a human immunodeficiency virus anti-sense mRNAsequence. This DNA sequence may comprise the DNA sequence which encodesa human immunodeficiency virus anti-sense mRNA sequence and is shown inFIG. 6.

Additionally the foreign DNA sequence may encode an antigenicpolypeptide. In one embodiment of the invention, the foreign DNAsequence encodes a malarial surface antigen. In another embodiment ofthe invention, the foreign DNA sequence encodes beta-galactosidase.

In still another embodiment of the invention, the foreign DNA sequenceis inserted into the human cytomegalovirus at the EcoRI restrictionendonuclease cleavage site of the 5.4 kb BamHI fragment shown in FIG. 1.In yet another embodiment of the invention, the foreign DNA sequence isinserted into the human cytomegalovirus so as to replace the 5.4 kb XhoIdeletion fragment of the EcoRI fragment shown in FIG. 2.

In another embodiment of the invention, the foreign DNA sequence isadapted for expression by an endogenous upstream human cytomegaloviruspromoter. The endogenous upstream human cytomegalovirus promoter may bethe immediate early promoter.

Furthermore the foreign DNA sequence may be adapted for expression by aheterologous upstream promoter. In one embodiment of the invention, theheterologous upstream promoter is the pseudorabies gpX promoter. Inanother embodiment of the invention, the heterologous upstream promoteris the human cytomegalovirus immediate early promoter.

The present invention also provides a plasmid designated pSY1157. Thisplasmid has been deposited with the American Type Culture Collection(ATCC) Rockville, Md. 20852, USA, in the E. coli host cell JM101 underATCC Accession No. 67452. Also provided by the present invention is aplasmid, designated pSY1159, which has been deposited with the ATCC inthe E. coli host cell JM101 under ATCC Accession No. 67453.

The human cytomegalovirus Towne strain, S-HCMV-001 has been depositedwith the ATCC under ATCC Accession No. VR 2174. These deposits were madeon Jun. 25, 1987.

The present invention also provides a therapeutic composition comprisingan attenuated virus including a DNA sequence essential for replicationof a human cytomegalovirus and a foreign DNA sequence which encodes ahuman immunodeficiency virus anti-sense mRNA sequence, and apharmaceutically acceptable carrier. Pharmaceutically acceptablecarriers are known in the art to which the present invention pertainsand include, but are not limited to, 0.01-0.1M, preferably 0.05M,phosphate buffer or 0.8% saline. This therapeutic composition may beused to treat a subject infected with human immunodeficiency virus byadministering to the subject an effective human immunodeficiency virustreating amount of the therapeutic composition provided herein.

Also provided by the present invention is a vaccine comprising anattenuated virus including a DNA sequence essential for replication of ahuman cytomegalovirus and at least one foreign DNA sequence adapted forexpression of an antigenic polypeptide, and a pharmaceuticallyacceptable carrier. This vaccine may be used to immunize a human subjectagainst a disease by administering to the subject an effectiveimmunizing amount of the vaccine provided herein.

In one embodiment of the invention, the virus of the vaccine includes aforeign DNA sequence which encodes a malarial surface antigen. Thisvaccine may be used to immunize a human subject against malaria.

The viruses, therapeutic compositions, vaccines and methods forimmunizing and treating human subjects provided by the present inventionare based upon the ability to construct novel viruses from wild type orattenuated human cytomegaloviruses. These novel viruses and methods fortheir preparation will be better understood by reference to thefollowing experiments and examples which are provided for purposes ofillustration and are not to be construed as in any way limiting thescope of the present invention, which is defined by the claims appendedhereto.

EXAMPLES Example 1 I. Propagation of HCMV in Human Fibroblasts

a. Human Fibroblast Cultures

Fibroblasts from human embryonic lung and foreskins were compared fortheir ability to support the growth of the Towne strain of HCMV. We haveexamined W138 cells (human embryonic lung, passage 24); Flow 5000 (humanembryonic fibroblasts, passage 16); Flow 7000 (human foreskinfibroblasts, passage 17); and EC100 cells (human foreskin fibroblasts,passage 10). Due to the superior quality of HCMV plaques on foreskinfibroblasts (HFF) and the lower passage number of the available cells,HFF cells were chosen for further work. Flow 5000, Flow 7000, EC100, orprimary cultures prepared in our lab all gave satisfactory results. Flow7000 cells were obtained from Flow Laboratories at passage 17 and usedfor most of the experiments reported here, although recently we havebegun to use EC100 cells which have a lower passage number. The cellsare routinely cultured in minimal essential medium (MEM) containing 10%fetal bovine serum, 2 mM L-glutamine, and 100 units/ml of pen-strep.Cultures are split 1:2 by trypsinization and resuspension in freshmedium. We refer to the various human foreskin fibroblast cells as HFFregardless of commercial origin as they are essentially interchangeable.

b. Infection with HCMV

HFF are infected when they reach 80% confluence at a multiplicity ofinfection (MOI) of 0.01 to prevent the formation of defectiveinterfering particles. When the cells demonstrate 100% cytopathiceffect, they are harvested, centrifuged and resuspended in a 1:1 mixtureof growth medium and sterile skim milk (9% w/v dry skim milk in water).The cells are then sonicated on ice and stored at -70° C. We routinelyobtain titers of 10⁸ PFU/ml for Towne strain by this method. StrainAD169, which was provided by ATCC grows more slowly than Towne andreached titers of 10⁷ PFU/ml. A clinical isolate of HCMV, strain"Jerry", was obtained from Dr. Jay Nelson, Scripps Clinic and ResearchFoundation, La Jolla, Calif. This virus grows more slowly than Towne orAD169 and gives titers of 10⁶ PFU/ml.

c. Plaque Assay for HCMV

HFF are plated in 6-well Falcon dishes at 80% confluency. The next day,virus stocks are diluted 1:10⁵, 10⁶, and 10⁷ in growth medium. Fromthese diluted samples, 10 microliters are added to each well containing1.5 ml of fresh medium containing 5% fetal bovine serum (FBS). Cellmonolayers are examined with a microscope for the appearance of plaquesconsisting of rounded cells. For the Towne strain, these plaques can bequantitated 5-6 days post infection. For AD169, 10-12 days were requiredto see plaques, and for "Jerry", 12-14 days are necessary to distinguishplaques. Quantifiable dishes contain between 20 and 50 plaques.

d. Transfection with HCMV DNA

We use various methods to transfect viral DNA from a number of animalherpesviruses. We have tested these various methods to transfect HCMVDNA. It is our observation that HCMV DNA can transfect HFF cells byCaCl₂ /DMSO and Polybrene/DMSO treatment but does not transfect by DEAEdextran/DMSO, CaCl₂ /glycerol, nor CaCl₂ /PEG treatments. In all cases,HCMV DNA was not CsCl purified and the DNA was transfected into HFFcells. As a control, pseudorabies virus DNA was also successfullytransfected into HFF cells by CaCl₂ /DMSO treatment but did nottransfect by CaCl₂ /glycerol, nor DEAE dextran/DMSO treatments.

We have compared the transfection assay in human fibroblast cells atvarious passage levels. For example, using the Polybrene/DMSO method, weobserved plaques in Flow 5000 cells on day 10 post-transfection andplaques in Flow 7000 cells on day 6 post-transfection. Flow 7000 cellsappear to produce plaques earlier than the Flow 5000 cells. In addition,the plaques in Flow 7000 cells are more compact. Because the plaques aremore distinct, they are more easily picked. Therefore, lower passage HFFcells were chosen to use when transfecting HCMV DNA and screeningrecombinant viruses.

A transient expression system was used to determine that specific geneconstructions were functional (see Section 1.4). In these assays, it waspossible to use a standard transfection protocol to introduce plasmidDNA into HFF cells ((25), Section 1.4). However, when HCMV recombinantswere produced by homologous recombination between plasmids and viralDNA, the polybrene method mentioned above was necessary in order toobtain satisfactory results using the much larger HCMV DNA. In thisprocedure, HFF cells were plated into 60 mM dishes at 80% confluency andallowed to attach. The cells were pre-treated with 4 micrograms/mlpolybrene in growth medium overnight (18-20 hrs) at 37° C. This mediumwas removed and replaced with 1 ml of growth medium containing 12micrograms/mi polybrene and the appropriate amount of HCMV DNA (seebelow). After adsorbing the DNA to the cells for 30 min. in the 37° C.CO₂ incubator, growth medium was added without polybrene and incubatedan additional 3.5 hrs. The inoculum was replaced with 30% DMSO in Hank'sBalanced Salt Solution (HBSS) for 4 min. at room temperature and thenthe cells were washed once with HBSS, fresh growth medium was added andincubation continued overnight. The following day the cells were re-fedwith fresh growth medium and incubated until plaques formed.

Optimization of DNA Concentration: After titering many stocks of HCMVDNA, we established that 1-2 micrograms of HCMV DNA is the optimum forour transfection assays.

With the polybrene/DMSO transfection experiments, we routinely obtained1-15 plaques/1-2 micrograms HCMV DNA for each HCMV DNA preparation andsaw no increase in the number of plaques by further increasing theamount of HCMV DNA.

Example 2 Infection of OKT4⁺ Lymphocytes with HCMV

The AIDS virus, HIV, has been shown to infect OKT4⁺ helper T-lymphocytes(23). It is thought that this infection leads to the decrease in T4lymphocytes observed in AIDS patients and is at least in partresponsible for the immunodeficiency caused by this virus. The successof our strategy to deliver an AIDS anti-sense message with HCMV dependsupon the ability of HCMV to infect T4 lymphocytes or other cell typespermissive for HIV replication and to express the anti-sense message.The feasibility of this approach depends upon the ability of arecombinant HCMV to express a foreign gene in HIV permissive cells.

a. OKT4 Cell Culture

For the present experiments we have used HUT-78 cells (ATCC TIB161) asthe prototype OKT4 cells. These cells grow in suspension and aremaintained in RPMI medium containing 10% heat inactivated fetal bovineserum, 2 mM glutamine, and 100 units/ml pen-strep. We routinely usevolumes of 20 mls contained in T-75 flasks standing on end. Since thesecells condition their medium with Interleukin-2 (IL-2) (24), and otherlymphokines, they are split about 1:3 to approximately 2×10⁵ cells/mlevery 2 days by dilution with fresh medium without centrifugation of thecells. They routinely reach densities approaching 10⁷ cells/ml underthese conditions.

Frozen stocks were established by centrifuging the cells, resuspendingthem to about 5×10⁶ cells/ml and 1 ml per ampule in RPMI containing 20%fetal calf serum, 2 mM glutamine, 100 units/ml pen-strep and 5% DMSO,freezing overnight at -70° C. and storage in liquid nitrogen.

b. HCMV Infection

HUT-78 cells were cultured in RPMI medium containing 10% heatinactivated fetal bovine serum, 2 mM glutamine, 100 units/ml ofpen-strep and 10 one-half maximal units/ml of IL-2. After six days inculture, the cells are pelleted by centrifugation and resuspended in asufficient volume of stock virus prepared in HFF cells, so that themultiplicity of infection is 5. The mixture is then incubated at 37° C.for 3 hrs., diluted with RPMI, and the cells pelleted by centrifugation.The cells are then re-fed with fresh RPMI medium containing 10 one-halfmaximal units of IL-2/ml or with a mixture of that medium and 50% mediumpreviously conditioned by uninfected OKT4 cells.

Three assays were used to monitor the HCMV infection of HUT-78 cells:

1. Determination of beta-galactosidase activity following infection withthe HCMV Towne strain containing the E. coli beta-gal gene. At varioustimes after infection an aliquot of the infected culture is removed,centrifuged and beta-gal activity determined directly on the supernateand on the cleared cellular lysates. The cells were disrupted with 1%NP-40 in PBS and centrifuged to produce the lysates.

2. Determination of cell free virus titer. Infected HUT-78 cells arecentrifuged and the virus titer in the supernate determined by plating5, 50, 500 lambda aliquots in HFF cells. Plaques are quantitated byexamination with a microscope. Results are expressed as virus titer per1×10⁵ HUT-78 cells.

3. Infectious center assay. This assay was used to determine thepercentage of HUT-78 cells containing infectious virus. At various timesafter infection cells were removed, washed three times by centrifugationin fresh medium and resuspended in 1 ml of medium. Dilutions of 1:10¹,10², 10³, 10⁴ were made in medium and added to a suspension of HFF cells(1×10⁵) which results in approximately 80% confluency when plated intoFalcon 6-well dishes. The cells were mixed and plated. The cells wereallowed to settle overnight. The next day, the medium was carefullyremoved and the cultures overlaid with 0.75% low-melting temperatureagarose in medium MEM containing 5% fetal bovine serum, 2 mM glutamine,and 100 units/ml pen-strep. Cultures were incubated and examined forplaques. In the case of the beta-gal containing HCMV infections, blueplaques were determined following the addition of Bluogal as substrate.

Example 3 Deletion of Regions of HCMV to Better Attenuate the Virus andLocalize Positions for Insertion.

One of the primary objectives is to insert and express a gene coding forHIV anti-sense RNA into HCMV. We have identified sites on the HCMVgenome where deletions and/or insertions of foreign genes can be madeand which permit expression of foreign genes.

Construction of 9 HCMV Clones: Summary: A series of 9 regions of theHCMV genome were cloned into plasmid vectors in E. coli. Attempts weremade to insert the marker enzyme E. coli beta-galactosidase (beta-gal)into the cloned HCMV DNA. To date, 2 constructions carrying the beta-galinsert have been made and confirmed (starred in tabulation presentedbelow).

As diagrammed in FIG. 1, three larger fragments from repeat regions andthe long unique regions of HCMV (Towne) were isolated and cloned. Fromthese 3 larger fragments, smaller fragments were subcloned and used forinsertion of beta-gal. Listed below are the larger cloned fragments andthe subclones derived from these fragments. The size of the cloned HCMVfragment and the site of insertion is described.

1. Xba E Fragment, 19 kb, part of the long unique region

Five fragments were sub-cloned from the 19 kb Xba E fragment.

    ______________________________________                                        SACI B       5.5 kb    insert at the SalI site                                BamHI B      5.2 kb    insert at the SalI site                                BamHI A      7.5 kb    insert at the SalI site                                sub-clone of SAC C     insert at the SalI site                                BamHI A:                                                                      sub-clone of Pst C     insert at the SalI site                                SAC C:                                                                        ______________________________________                                    

2. Xba O Fragment: 4.8 kb, long internal repeat Insertion at MLUI site.

3. Xba D Fragment: 19 kb, long internal repeat, short internal repeatand part of unique short

Three fragments were subcloned from the Xba D fragment:

    ______________________________________                                        BamHI*        5.4 kb   insert at RI site                                                             (successful insertion)                                 BamHI          10 kb   insert at RI site                                      EcoRI H*     10.2 kb   insert into 5.4 kb XhoI                                                       deletion (successful                                                          insertion)                                             ______________________________________                                    

At the present time, we have the above 9 cloned fragments available forbeta-gal insertion. Beta-gal was placed into the RI site of the 5.4 kbBamHI fragment from Xba D and into the 5.4 kb XhoI deletion from theEcoRI H fragment (10.2 kb) derived also from Xba D. These constructionsare further detailed.

1.1 Construction of a homology vector, pSY920, for insertion into EcoRIH fragment using Pseudorabies gpX promoter:

The promoter of pseudorabies glycoprotein X (gpX) provided a convenientand active promoter to fuse to beta-gal. We have had some experiencewith this promoter which speeded the genetic constructions and whichgave us confidence that it might function well in a heterologous system.

1.2 The beta-gal construction

Summary: The coding region of the glycoprotein X (gpX) gene ofpseudorabies was deleted and replaced by the structural gene for E. colibeta-galactosidase. The resulting construct places beta-gal under thepseudorabies promoter control with the gpX poly A and terminationsignals. This construction is inserted into a derivative of pSP64downstream from the SP6 promoter at the SalI site.

Constructions: The promoter for pseudorabies gpX lies in the BamHI #10fragment and the bulk of the gene extends into the BamHI #7 fragment ofthe pseudorabies virus (PRV) genome. Both BamHI #10 and BamHI #7 werecloned and the BamHI #7 fragment was cut at the NdeI site, removingalmost all of the coding region of gpX to the C terminus. The NdeI sitewas filled in. The BamHI to BalI fragment of the E. coli beta-gal genewas inserted at the BamHI site of the gpX promoter fragment (BamHI #10)and the C terminal of gpX (Nde site of BamHI #7). The resultingconstruction fuses the gpX promoter and translational start in-frame tothe beta-gal structural gene and provides the gpX termination and poly Asignals. The entire construct resides on a 4.4 kb SalI fragment whichwas cloned into the multicloning site of pSP64 downstream from the SP6promoter region to form plasmid pSY844.

1.3 Insertion of PRV gpX/beta-gal into the EcoRI H fragment at the Xho Ideletion

The EcoRI H fragment (10.2 kb) includes part of the long internalrepeat, the junction region, the entire short internal repeat and partof the unique short region (FIG. 1). This fragment was cloned and twointernal XhoI fragments totalling 5.4 kb were removed. The deletedfragment includes part of the long internal repeat, the junction region,the short internal repeat and a fraction of the unique short region. The4.4 kb SalI fragment carrying beta-gal (see 1.2 above) was ligated intothe Xho I deletion site. The resulting construction flanks the beta-galgene with HCMV sequences appropriate for insertion by homologousrecombination into the HCMV virus (Homology vector, pSY920) (see FIG.2).

1.4 Confirmation of a functional construction of the homology vectorpSY920 by transient expression

In order to determine whether the HCMV/beta-gal homology vectorconstruction was correct and would function when inserted into HCMV,beta-gal expression was tested in a transient expression experiment. Inthis experiment the homology vector plasmid DNA is introduced into cellsfollowed by superinfection with wild-type HCMV. The viral transactingregulatory signals should activate and allow for beta-gal expressionfrom the plasmid DNA if the beta-gal construction is correct and if thegpX promoter responds to the heterologous HCMV signals.

HFF cells were transfected with plasmid DNA according to the method ofSpaete and Mocarski (25). 10 micrograms DNA were suspended in MEM media(plus 100 units/ml penicillin and streptomycin, each 2 mM glutamine, noserum and 200 micrograms/ml DEAE Dextran). DNA was allowed to absorb for4 hours, the medium was removed and cells were re-fed with the samemedia, without DEAE Dextran, containing 10% fetal calf serum. 16 hourslater the cells were again re-fed. At 48 hours after transfection, thecells were infected at a multiplicity of 10 with HCMV. 10 hours later,the cells were harvested and tested for beta-gal.

Beta-gal assay of infected monolayers: The infected cell monolayers (60mM dish) were washed in phosphate buffered saline (PBS) and lysed in 0.2ml PBS containing 1% sodium dodecylsulfate (SDS). The lysate (200microliters) was diluted into Z buffer (800 microliters) (35), theenzyme substrate (ONPG, 200 microliters) was added and the developmentof yellow color was monitored at 420 nm (37° C.). As shown in Table 1,pSY920 produced beta-gal in this transient expression test indicatingthat the construction was correct and functional.

                  TABLE 1                                                         ______________________________________                                        TRANSIENT EXPRESSION ASSAY                                                    β-GAL ACTIVITY IN LYSATES                                                                      Intensity of                                            Plasmid    HCMV       Enzyme Response                                                                            O.D. 420                                   ______________________________________                                        pSY920     +          ++++         2.848                                      PRV gpX/β-gal                                                                       -          -            0.218                                      HCMV EcoRI H                                                                  pSY921     +          +++          2.024                                      HCMV IE/β-gal                                                                       -          +/-          0.286                                      No plasmid +          -            0.228                                                 -          -            0.212                                      ______________________________________                                    

These results also indicate that the heterologous gpX promoter frompseudorabies is functional in HFF cells and responds well to HCMVregulatory signals. In similar experiments, pSY920 also expressesbeta-gal transiently in response to pseudorabies super-infection, aswould be expected (Table 2).

                  TABLE 2                                                         ______________________________________                                        TRANSIENT EXPRESSION ASSAY                                                    β-GAL ACTIVITY IN LYSATES                                                                       Intensity of                                           Plasmid    Pseudorabies                                                                              Enzyme Response                                                                            O.D. 420                                  ______________________________________                                        pSY920     +           ++           1.343                                     PRV gpX/β-gal                                                                       -           -            0.218                                     pSY921     +           +            0.961                                     HCMV IE/β-gal                                                                       -           +/-          0.286                                     No plasmid +           -            0.228                                                -           -            0.212                                     ______________________________________                                    

1.5 Insertion of PRV gpX/beta-gal into HCMV using pSY920

pSY920 plasmid DNA was combined with HCMV DNA and transfected into HFFcells using the polybrene protocol (Section Id).

In this case a positive stock was selected and plated out. A total of 76blue plaques were picked of which 58 (76%) gave rise to secondary viralstocks. Of these secondary stocks, 5 (8.6%) were positive by ONPG assayof the supernatants from infected cell cultures. The recombinant viruswas further purified through additional rounds of plaque picking.

1.6 Confirmation of recombinant HCMV

The presence and location of the beta-gal gene inserted into HCMV wasconfirmed by Southern blot analysis. FIG. 3B presents the data fromanalysis of this recombinant virus containing beta-gal as an insert intothe Xho deletion. Lane 1 contains molecular weight markers. Lane 2contains plasmid pSY1112 DNA digested with EcoRI and used as a controlfor the PRV gpx/beta-gal gene. Two fragments of 8.2 kb and 4.4 kbrepresenting both the plasmid sequences and the beta-gal generespectively are revealed. Lane 3 contains Towne DNA digested with EcoRIand probed with beta-gal DNA demonstrating that there is nohybridization of the probe to the parent virus. Lane 4 contains DNA fromthe recombinant virus probed with beta-gal and reveals two fragments Hand U that contain he inserted E. coli gene. We interpret this result toindicate that homologous recombination has occurred in both the internaljunction fragment and one of the terminal repeat fragments that containsthe same DNA sequence.

These results indicate that it is possible to delete the Xho region ofEcoRI H and to insert and express foreign genes at this point on theHCMV genome. This region is probably permissive for such manipulationsdue to the presence in the virus genome of an unaltered duplicate of theinformation contained in this region.

2.1 Construction of a second beta-gal construction, pSY921, forinsertion into EcoRI H fragment using CMV IE promoter

Our goal is to express a gene coding for anti-sense RNA for HIV in OKT4lymphocytes and other cells permissive for HIV. HCMV infects OKT4lymphocytes and expresses at least immediate early (IE) genes in thesecells (26, 27) and probably replicates as well (28). It was importantfor our objectives to express a foreign gene under the control of theHCMV IE Promoter.

2.2 Beta-gal construction

Summary: The immediate early promoter region of HCMV was cloned andfused to a beta-gal construct which contained the HSV TK polyadenylation signal at the 3' end.

Constructions: we have cloned the 19 kb XbaI E fragment from the longunique region. This fragment contains the IE promoter region (29). A 2.1kb Pst fragment was sub-cloned from the XbaI piece (see FIG. 4). The Pstfragment includes the IE promoter and enhancer, cap site, the first exonand the first 2 introns up to the AUG protein start.

This enhancer/promoter fragment was fused to a beta-gal fragmentcontaining the following elements. The beta-gal enzyme is truncated atthe 5' end at the BamHI site located 4 amino acids from theamino-terminus. Located at the 5' end of this gene is the promoterregion of the HSV TK gene including the protein start codon of TK whichis fused in-frame to the beta-gal gene. The Pst-BamHI fragment from the3' end of the HSV TK gene carrying the poly adenylation signal islocated at the 3' end of the beta-gal gene. The IE promoter Pst fragmentwas joined to the 5' end of this beta-gal construct at the Pst site ofthe HSV TK region replacing the HSV TK promoter with the HCMV IEpromoter to create pSY921.

2.3 Insertion of HCMV-IE/beta-gal (pSY921) into EcoRI H fragment at XhoI deletion (pSY930)

As shown in FIG. 2, the 6.1 kb HCMV IE/beta-gal construction wasinserted into the 5.4 kb Xho I deletion region of the EcoRI H (10.2 kb)fragment (see Section 1.3). This construction is pSY930.

2.4 Confirmation of a functional construction of the pSY921 by transientexpression

As described in Section 1.4, the DNA from the HCMV IE/beta-gal plasmidpSY921 was tested in the transient expression system. The plasmidbeta-gal gene responded to HCMV signals and expressed the enzyme. Inthis construction, the IE promoter may produce less enzyme than the gpXpromoter in the transient assay system (Tables 1, 2). However, thebeta-gal expression was observed to begin sooner after super-infectionthan did gpX promoted expression. In addition, there was some measurableexpression of the beta-gal from the IE promoter in the absence of viralsuper-infection (see Table 1).

It is interesting to note that pSY921 also expressed beta-gal in thissystem when pseudorabies was used to super-infect the cells (Table 2).Thus, just as the gpX promoter responded to heterologous regulatorysignals when located in HCMV, the IE promoter of HCMV responds to theheterologous signals of pseudorabies.

These results indicated that pSY921 was intact and functional.

2.5 Insertion of HCMV/beta-gal IE into HCMV at EcoRI H using pSY930

pSY930 DNA was combined with HCMV DNA and transfected into HFF cells asdescribed in Section 1.5. A beta-gal positive transfection stock wasidentified and plated out. Twenty-seven positive plaques were pickedfrom this plating for further purification. Work continues on thefurther purification of this recombinant virus. However, the preliminarypositive results indicate that the IE promoter probably functions toexpress foreign genes in the EcoRI H location in HCMV. Thus foreigngenes can be expressed at this site in HCMV either under control ofhomologous (IE) or heterologous (gpX) herpesvirus promoters.

3.1 Identification of a second region of HCMV permissive for insertionand expression of foreign genes

We are continuing to examine other regions of the HCMV genome inaddition to the Xho junction fragment for the insertion and expressionof foreign genes. Located within the 19 kb Xba D fragment is a 5.4 kbBamHI fragment containing a portion of the long internal repeat. Thisfragment expresses 2 mRNA's: a 1.2 kb early message and a major 2.7 kbearly message. The 2.7 kb mRNA is the most abundant mRNA produced atboth early and late times during infection and maps to about 600basepairs upstream of the RI site on this fragment (29).

3.2 Construction of the homology vector pSY1112

This cloned 5.4 kb BamHI fragment was opened at the RI site and the PRVgpx/beta-gal construct (described above (1.2) was inserted (see FIG. 5).As the integrity of the beta-gal construct had already been established,this new plasmid (pSY1112) construct was not further tested by transientinfection but was used directly in homologous recombination.

3.3 Insertion of PRV gpX/beta-gal into HCMV BamHI (5.4 kb) using pSY1112

A number of independent transfection stocks were made in which pSY1112and HCMV DNA were mixed in various proportions and transfected in HFFcells using the polybrene method. The results of these transfections aredescribed in Table 3. One stock out of 30 showed higher than expectedbeta-gal activity (CT3-3) and this was used for the isolation ofrecombinant virus. A recombinant HCMV was purified from this stock byplaque purification. This recombinant proved to give the best yield ofvirus and enzyme among the HCMV recombinants constructed to date. As aresult, our initial efforts have been focused on this site for theinsertion of foreign genes. This recombinant virus was designatedS-HCMV-001. Stocks of this virus typically produced a low level (about1%) of white plaques when assayed for beta-gal production. These whiteplaques have not been characterized.

                  TABLE 3                                                         ______________________________________                                                   HCMV    Day post CT                                                Stock  Plasmid   DNA       plagues                                                                             harvest                                                                              ONPG                                  ______________________________________                                        CT3-1  1112 0.5 μg                                                                          10-20/    14    31     -                                     2                (10/3)    22    32(D)  ND                                    3                           31*  37     ++++                                  4                          14    31     -                                     5         1.0 μg        -     32(D)  ND                                    6                          22    35     -                                     7                           31*  32     -                                     8                          -     32(D)  ND                                    9                no HCMV   -     32(D)  ND                                    10     no plasmid                                                                              no HCMV   -     32(D)  ND                                    CT4-1  1112 1.0 μg                                                                          15/       13    20     -                                     2                (10/3)    13    20     -                                     3                          13    20     -                                     4                          13    20     +                                     5                          13    17     -                                     6                          13    20     +                                     CT5-1  1112 0.5 μg                                                                          20/        8    16     +                                     2                (10/3)     8    18     -                                     3                           8    18     -                                     4                           8    18     -                                     5                (12/16)   12    21     -                                     6                           8    21     +                                     7                           8    21     -                                     8                          12    21     +/-                                   9      no plasmid          --    16(D)  ND                                    CT6-1  1112 0.5 μg                                                                          5/        11    15     +/-                                   2                (12/16)   11    20     -                                     3                          -     15(D)  ND                                    4                10/       11    20     -                                     5                           7    15     +/-                                   6                          11    15     +/-                                   7                20/        7    20     +/-                                   8                          11    18     ++                                    9      no pasmid no HCMV   --    15     ND                                    ______________________________________                                    

3.4 Confirmation of S-HCMV-001 structure by Southern blot analysis

In FIG. 3A viral DNA was digested with HindIII and probed in a Southernblot with the homologous XbaI M fragment (see FIG. 1 for restrictionmap) or with beta-gal DNA. When probed with the XbaI M fragment, HindIIIfragments E, G, and K from Towne DNA hybridize (lane 2). In therecombinant, both fragments E and G which result from inversion of theunique short region have increased in size due to the insertion of thebeta-gal gene, but the K fragment remains the same size (lane 3 and 1/2of lane 4). This indicates that the insertion of beta-gal in thejunction region in effect freezes the unique long segment of the genomein one orientation and prevents the normal reorganization of the genomethat occurs in this region. When the same digested DNA is probed withthe beta-gal gene (1/2 of lane 4 and lane 5), only fragments E and Ghybridize, confirming the conclusion reached above. Lane 6 shows thatthere is no hyridization of beta-gal with the Towne strain DNA asexpected.

Summary:

We have identified 2 sites on the CMV genome within the internal repeatregion which allow deletion and/or insertion of foreign genes and permitthe expression of these genes using either heterologous or homologousherpesvirus promoters.

Example 4 Design and Chemical Synthesis of DNA Coding for HIV Anti-senseRNA

Zamecnik, et al. (30) published a study on the effects of anti-sense RNAon HIV replication in vitro. This work pointed to specific anti-senseRNA's that were effective in blocking HIV replication. Based upon thisinformation, we designed the anti-sense gene. The synthesis of theanti-sense gene is outlined in Table 4.

4.1 Synthesis of DNA oligomers

The two fragments shown in Table 4 were synthesized as flows, usingconventional chemical synthesis methods:

                                      TABLE 4                                     __________________________________________________________________________    Oligomers Synthesized: (5' to 3')                                             1-A AATTGTCGACGCGTACTCACCAGTCGCCGCACACCCAATTCTGAAAATGGTCGAC                   1-B AGCTGTCGACCATTTTCAGAATTGGGTGTGCGGCGACTGGTGAGTACGCGTCGAC                   Final Fragments:                                                              Fragment A                                                                     ##STR1##                                                                     __________________________________________________________________________

The N,N-diisopropylphosphoramidites, controlled-pore glass columns andall synthesis reagents were obtained from Applied Biosystems, FosterCity, Calif.

Synthetic oligonucleotides were prepared by the phosphite triestermethod with an Applied Biosystems Model 380A DNA synthesizer using a10-fold excess of protected phosphoramidites and 1 micromole ofnucleotide bound to the synthesis support column. The chemistries usedfor synthesis are the standard protocols recommended for use with thesynthesizer and have been described (31). Deprotection and cleavage ofthe oligomers from the solid support were performed according tostandard procedures as described by McBride (32). The repetitive yieldof the synthesis as measured by the optical density of the removedprotecting group as recommended by Applied Biosystems (1984) was greaterthan 97.5%.

The crude oligonucleotide mixture was purified by preparative gelelectrophoresis as described by the Applied Biosystems protocols of Nov.9, 1984 (User Bulletin No. 13). The acrylamide gel concentration variedfrom 10 to 20% depending upon the length of the oligomer. The purifiedoligomer was identified by UV shadowing, excised from the gel andextracted by the crush and soak procedure (33).

The fragments 1-A and 1-B were mixed (50 ng of each) heated to 65° C.and allowed to anneal by slowly cooling at room temperature (10 min at55° C., 10 min at 47° C., 10 min at 42° C., 10 min at 37° C., and 10 minat room temperature). The annealed DNA was ligated into vector PBS M13+(from Stratagene) cut with EcoRI and HindIII and transformed into JM109.Colonies were screened by colony hybridization using P³² labeledfragment 1-A. Thirty positive clones were selected, DNA was isolatedfrom the colonies (mini prep method) and examined by agarose gelelectrophoresis for inserts. The DNA inserts of 6 plasmids weresequenced by the primer extension method for double stranded DNA's (34).Clone BSI-32 (pSY1157) was shown to contain the correct A fragmentsequence and was selected for further manipulation.

The A fragment was engineered for ultimate placement into an HCMVhomology vector in such a way that its expression would be controlled bythe HCMV IE promoter and the presence of the inserted gene could bedetected in HCMV by expression of beta-gal. The initial constructsinvolved placing fragment A downstream from the IE HCMV promoter andupstream of beta-gal so that anti-sense RNA synthesis would occur whenbeta-gal expression was observed. The first step in this procedure wasto construct and test an improved new HCMV IE promoter-beta-gal vector(pSY1132).

4.2 Construction of pSY1132

The PstI fragment containing the HCMV IE promoter enhancer region (FIG.4 and Section 2.2) was cut at the internal SacI site to create a 1.1 kbfragment carrying the IE promoter and enhancer and part of the firstintron, including the cap site. This shortened IE promoter fragment wasthen linked to the beta-gal gene using a fragment of herpes simplexthymidine kinase (HSV TK) gene to supply the untranslated 5' end of themRNA. The RsaI fragment at the 5' end of HSV TK was coupled to BamHIlinkers and joined to the 5' end of the BamHI beta-gal fragment(described above, Section 2.2). This construction contains 6 additionalamino acids, 3 from the 5' end of the TK structural gene and 3 from theBamHI linker (see FIG. 6). The beta-gal carries the HSV TK genepolyadenylation signals at the 3' as described (Section 2.2). ThePst-Sac IE HCMV fragment is inserted at the cap site of the HSV TKsegment (FIG. 6) to create pSY1132.

4.3 Confirmation of a functional construction by transient expression

pSY1132 was tested in the transient expression system (Table 5). PlasmidpSY844 (see Section 1.1) served as a positive control in these assays.

                  TABLE 5                                                         ______________________________________                                        Transient Expression of the AIDS Anti-sense Sequence                                                 Intensity of                                           Plasmid       HCMV     Enzyme Response                                                                            O.D. 420                                  ______________________________________                                        pSY1132       +        ++++         2.8301                                    HCMV IE/β-gal                                                                          +        ++++         2.8247                                                  +        ++++         2.8219                                                  -        +/-          0.1049                                    pSY844        +        ++           0.8621                                    PRV gpX/β-gal                                                                          +        ++           0.7319                                                  +        ++           1.0014                                                  -        -            -0.0481                                   pSY1159       +        +            0.2384                                    HCMV IE/AIDS/β-gal                                                                     +        +            0.1816                                                  +        +            0.2026                                                  -        -            -0.0529                                   pSY1160       +        +/-          0.1166                                    HCMV/IE/AIDS/β-gal                                                                     +        +/-          0.1469                                                  +        +/-          0.1457                                                  -        -            -0.0036                                   No plasmid    +        -            -0.0211                                                 -        -            -0.0460                                   ______________________________________                                    

Infected cell monolayers were washed in PBS and lysed in PBS containing1% non-ionic detergent (NP40). An aliquot of the lysate (100microliters) was diluted into (700 lambda) Z buffer and assayed asdescribed previously.

The results from these assays indicate that pSY1132 is intact andfunctional. In addition, this construct with the truncated HCMV IEpromoter/enhancer fragment is the most active of the herpes promoterbeta-gal fusions so far tested (compare to pSY844). As in the case ofpSY921 (carrying the larger HCMV IE fragment), the plasmid DNA wascapable of some slight beta-gal expression in the absence of CMVsuper-infection.

4.4 Construction of anti-sense RNA gene: beta-gal fusion vectors(pSY1159 and 1160)

The initial construction of the anti-sense homology vector involvedinsertion of the anti-sense gene from pSY1157 (see Section 4.1) into theBglII site preceding the beta-gal gene in pSY1132. In order to achievethis the fragment A construction was cut from pSY1157 with SalI and the45 basepair Sal insert was purified from a 3% low melting point agarosegel. The vector pSP72 (a derivative of pSP64) was cut with SalI in themultiple cloning site downstream of the SP6 promoter and was ligated tothe purified A fragment. This construction (pSY1158) places theanti-sense RNA gene into a multiple cloning site with unique BamHI sitesflanking the construct. The insert was then cut from the plasmid withBamHI and the purified insert DNA, now with BamHI ends, was ligated intothe BglII site of pSY1132 within the HSV TK region immediately upstreamfrom the beta-gal 5' end and downstream from the HCMV IEpromoter/enhancer. The insertion of the anti-sense fragment occurred in2 orientations, the "correct" anti-sense orientation relative to thepromoter (pSY1159) and in the "incorrect" sense orientation (pSY1160).The constructions pSY1159 and 1160 were confirmed by double-strandeddideoxy sequencing and by restriction analysis.

4.5 Effect of anti-sense gene insertion on beta-gal construction:transient expression assay

pSY1159 and 1160 were tested in the transient expression assay system(Table 5). When the anti-sense sequence is present in the (correct)anti-sense orientation (pSY1159), the downstream beta-gal gene isexpressed at low levels in the system. However, when the sequence is inthe opposite orientation at the same location (pSY1160), even lessbeta-gal is detected. It is not clear why the anti-sense sequenceaffects the expression level of beta-gal and work is under way toinvestigate this phenomenon.

4.6 Constructions to improve the level of anti-sense expression

pSY1159 and 1160 do produce measurable beta-gal in transient expression.However, before preceding to insert the anti-sense gene into HCMV betterexpression of the gene is desirable. We have shown that some genes orDNA sequences when fused to the N-terminal of beta-gal severely depressbeta-gal expression. When these same sequences are fused to theC-terminal of beta-gal, much higher levels of expression are permittedupon infection with the recombinant pseudorabies.

The AIDS anti-sense gene has been placed behind beta-gal as a carboxyfusion and the expression level has been raised dramatically.

Summary: Anti-sense RNA gene constructions:

We have constructed a 50 basepair anti-sense RNA gene from HIV basedupon published data indicating that this sequence is effective in vitroin blocking HIV replication. The anti-sense RNA gene has been placedunder the control of a strong IE HCMV promoter/enhancer sequence andbeta-gal expression has been engineered as a marker for anti-sense RNAexpression. With the anti-sense RNA fused to the carboxy terminis ofB-gal, the expression level of RNA and protein is good.

Example 5 Infection of HUT78 Cells in vitro with Recombinant HCMV andExpression of a Foreign Gene

In order to deliver the AIDS anti-sense RNA to target OKT4 cells usingour strategy, HCMV must be able to infect these cells and activelytranscribe RNA from the immediate early promoter. A recent report (28)describes the replication of HCMV AD169 in T-lymphocytes. Thedemonstration of infected cells and increases in viral titer werepresented as evidence for infection. These data appear to show that bothOKT4⁺ and OKT8⁺ subpopulations can support HCMV replication. Thepresence of immediate early transcripts from HCMV in infected OKT4⁺cells has also been demonstrated (26). In the experiments reported here,we have used recombinant HCMV containing the beta-gal cassette insertedat the RI site in the BamHI fragment of the Towne strain. This virus,designated Strain 001, was chosen because it could be easily detected byblue plaque assay or by beta-gal enzyme determinations. In this virusthe beta-gal gene is controlled by the PRV gpX beta-gal promoter asdescribed in Sections 3.1-3.4. We have also used Towne, AD169, and"Jerry" but with these viruses plaques must be quantitated by thestandard plaque assay. We have used three different assays to monitorthe infection of the OKT4 cell line HUT-78 with HCMV (as described inSection II, "Infection of OKT4⁺ Lymphocytes by HCMV").

The results of our experiments are shown in Table 6.

                  TABLE 6                                                         ______________________________________                                        Infection of OKT4.sup.+  Cells with HCMV                                      a) Infectious Center Assay (% Infectious Centers)                                     Days Post Infection                                                   Virus     3      5      7     10     12   14                                  ______________________________________                                        001       0.01   0.1    0.03  0.0    0.0  0.0                                 Towne     0.01   1.3    0.3   0.003  0.0  0.0                                 AD169     0.02   1.8    0.56  0.007  0.0  0.0                                 Jerry     0.02   0.0    0.0   0.0    0.0  0.0                                 no virus  0.0    0.0    0.0   0.0    0.0  0.0                                 ______________________________________                                         ##STR2##                                                                     b) Cell Free Virus Titer (PFU/10.sup.5 cells)                                         Days Post Infection                                                   Virus     4      6        8      11    14                                     ______________________________________                                        001       20     200      4.8    0     0                                      Towne     600    670      50     0     0                                      AD169     350    1300     200    6     0                                      Jerry     29     21       0      0     0                                      no virus  0      0        0      0     0                                      ______________________________________                                    

Both the infectious center assay (a) and the assay of cell free virus(b) indicate that HCMV is capable of infecting HUT-78 cells. Theinfectious center assay indicates that the maximum number of infectedOKT4 cells (maximum of 1.8% in this experiment) is observed 5-7 dayspost infection. AD169 leads to more infected cells than does Towne whichin turn leads to more than its recombinant, 001, containing the beta-galgene of E. coli. Strain "Jerry", the clinical isolate, failed to producesignificant infectious centers in this cell line. These data aresupported by the cell free virus titers produced from infected HUT-78cultures (Table 6b). The highest virus titers were observed in AD169infected cells about 1 week after infection. Lower titers were obtainedfor the Towne strain and even lower for 001. Titers for strain "Jerry"did not increase during the experiment.

Establishing the infection of various lymphocyte cell types by HCMV iscomplicated by the carryover of the inoculum virus prepared as a crudecell lysate. In our experiments, the crude cellular lysate contains notonly virus but considerable amounts of beta-gal activity. This activitywas sufficiently high and stable as to mask any activity produced byOKT4 cells infected with 001. The results presented in Table 5 shouldnot be affected by the carryover of material from the inoculum. Risingvirus titers and increasing infectious centers indicate activeinfection. In subsequent experiments we are using cell-free viruspreparations and are taking samples at zero time immediately afterinfection and absorption to better control any potential variables.

REFERENCES

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9. G. V. Quinnan Jr., et al., Ann. Intern. Med. 101:478-483, 1984.

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What is claimed:
 1. Recombinant cytomegalovirus comprising (i) thegenomic DNA of Towne strain human cytomegalovirus; (ii) a foreign DNAsequence inserted within the 5.4 Kb BamHI region shown in FIG. 1 orwithin the 5.4 Kb XhoI region shown in FIG. 2 of Towne strain humancytomegalovirus genome; and (iii) a promoter sequence attached to theforeign DNA at location upstream of the said foreign DNA, the promotersequence being the endogenous human cytomegalovirus major immediateearly promoter or a foreign herpesvirus promoter.
 2. A virus of claim 1,wherein the foreign DNA sequence encodes a human immunodeficiency virusanti-sense mRNA sequence.
 3. A virus of claim 2, wherein the foreign DNAsequence comprises the nucleotidesequence5'-AATTGTCGACGCGTACTCACCAGTCGCCGCACACCCAATTCTGAAAATGGTCGAC-3'3'-CAGCTGCGCATGAGTGGTCAGCGGCGTGTGGGTTAAGACTTTTACCAGCTGTCGA-5'.
 4. Avirus of claim 1, wherein the foreign DNA sequence encodes an antigenicpolypeptide.
 5. A virus of claim 4, wherein the foreign DNA sequenceencodes a malarial surface antigen.
 6. A virus of claim 1 wherein theforeign DNA sequence encodes beta-galactosidase.
 7. A virus of claim 1,wherein the foreign herpesvirus promoter is the pseudorabies gpXpromoter.
 8. A plasmid designated pSY1157.
 9. A plasmid designatedpSY1159.